Method for the suppression of visceral pain by regulating T type calcium channel

ABSTRACT

The disclosure concerns a method for the suppression of visceral pain by regulating the T-type calcium channel; a visceral pain inhibitor that includes a T-type calcium channel inhibitor as an effective ingredient; and a method of screening a visceral pain inhibitor by investigating the suppression activity of T-type calcium channels. Particularly, the present invention relates to a method for the suppression of visceral pain by regulating an alpha 1G T-type calcium channel in the central nervous system and alpha 1H and alpha 1I T-type calcium channels in the peripheral nervous system; a visceral pain inhibitor that includes a T-type calcium channel inhibitor as an effective ingredient; and a method of screening a visceral pain inhibitor by investigating the suppression activity of T-type calcium channels. The method of the present invention can be effectively used to suppress visceral pain by regulating T-type calcium channel in a precise mechanism without any side effects.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for the suppression ofvisceral pain by regulating the T-type calcium channel; a visceral paininhibitor that includes a T-type calcium channel inhibitor as aneffective ingredient; and a method of screening a visceral paininhibitor by investigating the suppression activity of T-type calciumchannels. Particularly, the present invention relates to a method forthe suppression of visceral pain by regulating an alpha 1G T-typecalcium channel in the central nervous system and alpha 1H and alpha 1IT-type calcium channels in the peripheral nervous system; a visceralpain inhibitor that includes a T-type calcium channel inhibitor as aneffective ingredient; and a method of screening a visceral paininhibitor by investigating the suppression activity of T-type calciumchannels.

BACKGROUND OF THE INVENTION

[0002] Visceral pain is usually caused by excessive dilation of internalorgans, necrosis of these cells or intensive contraction or acuterelaxation of internal organs. When there is a tumor, infection orcongestion in internal organs, slight mechanical stimulus, acidic orbasic solution might cause severe pain. Visceral pain caused especiallyby tumors cannot be easily suppressed even with an excessive amount ofmorphine, so neuro-surgical operations such as partial myelotomy of thespinal cord are frequently used (Gybels, Pain Headache, 1989, 11:1-402).However, the bilateral cordotomies or commissural myelotomies ofspinothalamic tract have many side effects. Relatively, the midlinemyelotomy that severs the upper middle part of the T10 spinal cord isknown as an effective remedy (Nauta, J. Neurosurg., 1997, 86:538-542).

[0003] The above result proves that the visceral pain signal isdelivered to the brain through the spinal cord, which supports the factthat the visceral pain signal is delivered through a different channelfrom other pains. According to a MRI test carried out ona-visceral-pain-induced monkey, it can be known that visceral paininduces the activation of thalamus (Willis, Proc. Natl. Acad. Sci. USA.,1999, 96:7675-79) The result of the test, after all, tells that thevisceral pain is delivered from the pain sensory cells in the end of theinternal organs through the spinal cord to thalamus. Particularly,thalamus is known as an important sensory processing organ since itdelivers the stimulus to the cerebral cortex (McCormick, Curr. Opin.Neurobiol., 1994, 4:550-556).

[0004] The calcium in nerve cells plays an important role in deliveringsignals between nerve cells. Calcium has many different delivery paths,however, when delivering peripheral stimuli, the voltage-activatedcalcium channel is crucial. The voltage-activated calcium channel can becategorized into the high voltage-activated calcium channel (HVA) thatis activated at a higher voltage than the resting membrane potential andthe low voltage-activated calcium channel (LVA) that is activated at alower voltage. The HVA calcium channel can be subdivided into L, P/Q, Nor R-type depending on the pharmacological property of the current, andthe LVA calcium channel is differentiated as T-type (Tsien, TrendsNeurosci., 1988, 11:431-438).

[0005] The HVA calcium channel is evenly expressed from the peripheralsensory cells to the central nervous system, and is well known to playan important role in transmission of the sense of pain and reflection.The inhibitors against these channels are already commercially availableas various anodynes (Schaible, Prog. Brain Res., 2000, 129:173-190).However, it is not yet clearly understood how the LVA calcium channelthat generates the T-type calcium current can regulate pain. The reasonwhy the T-type calcium current is categorized as one of the functions ofthe LVA calcium channel is that when the excitability of nerve cellslowers, the calcium current are generated so that the excitabilityincreases again (Llinas, J. Physiol (Lond), 1981, 315:549-567;McCormick, Neuroscience, 1990, 39:103-113). Thus, the nerve cellsexcited by the T-type calcium channel have the property of burst firingsand induce a type of excitability different from tonic firings (Llinas,J. Physiol (Lond), 1981, 315:549-567). The channel protein of the T-typecalcium channel is encoded by three different genes, which are referredto as alpha1G, alpha1H and alpha1I respectively (Perez-Reyes, Nature,1998, 391:896-900). It is known that the alpha1G and alpha1H T-typecalcium channels are expressed in the back of the spinal cord, and thatthe alpha1G is expressed in thalamocortical relay neurons (Talley, J.Neurosci., 1999, 19:1895-1911), and that is identical with the deliverypath of the visceral pain. Recently, it has been proved in an experimentusing a T-type calcium current inhibitor, mibefradil, that the functionof the T-type calcium current in the peripheral nerves is related tohyperalgesic reaction against thermo-stimuli or mechanical stimuli byreducing agents (Todorovic, Neuron, 2001, 31:75-85), however, it has notyet been found which T-type calcium channel is related. Mibefradil(RO40-5967) was initially known for lowering blood pressure (Clozel,Cardiovasc Drugs Ther., 1990, 4:731-736; Hefti, Arzneimittelforschung,1990, 40:417-421), and was reported to have a suppression effect (Viana,Cell Calcium, 1997, 22:299-311). Recently, it has been reported thatMibefradil has the most selective suppression effect on T-type calciumchannels.

[0006] Thus, the present inventors have studied about visceral pain withalpha1G−/− transgenic mice and found that the alpha1G−/− transgenic miceshow hyperalgesia to visceral pain caused by acetic acid. In wild-typemice, visceral pain caused by acetic acid could be alleviated byadministration of mibefradil at the periphery but enhanced whenmibefradil is injected in the brain. The present invention has beenaccomplished by confirming that visceral pain can be modulated bycontrolling the T-type calcium channel.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to provide a method forthe suppression of visceral pain by regulating the T-type calciumchannel.

[0008] It is another object of the present invention to provide avisceral pain inhibitor that includes a T-type calcium channel inhibitoras an effective ingredient.

[0009] It is a further object of the present invention to provide amethod of screening a visceral pain inhibitor by investigating thesuppression activity of T-type calcium channels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a graph showing the responses of alpha 1G−/− mice andnormal mice against mechanical stimuli such as paw withdrawal and tailflick test,

[0011] : normal mice ∘: alpha 1G−/− mice

[0012]FIG. 2 is a graph showing the responses of alpha 1G−/− mice andnormal mice against thermostimulus Hyperalgesia that is caused by athermostimulus by radiation or by inflammation,

[0013] ▪: normal mice □: alpha 1G−/− mice

[0014]FIG. 3 is a graph showing the responses of alpha 1G−/− mice andnormal mice against pain in internal organs that is caused by aceticacid,

[0015] : normal mice ∘: alpha 1G−/− mice

[0016]FIG. 4 is a graph showing the suppression degree in accordancewith the concentration of a T-type calcium channel inhibitor,mibefradil.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] The present invention provides a method for the suppression ofvisceral pain by regulating the T-type calcium channel.

[0018] The present invention also provides a visceral pain inhibitorthat includes a T-type calcium channel inhibitor as an effectiveingredient.

[0019] The present invention also provides a method of screening avisceral pain inhibitor by investigating the suppression activity ofT-type calcium channels.

[0020] Hereinafter, the present invention is described in detail.

[0021] The present invention provides a method for the suppression ofvisceral pain by regulating the T-type calcium channel.

[0022] The present invention provides a method for the suppression ofvisceral pain either by activating alpha 1G T-type calcium channelfunction in the brain or by suppressing alpha 1H and alpha 1I T-typecalcium channel function in the peripheral nervous system.

[0023] The T-type calcium channel is categorized into alpha 1G, alpha 1Hand alpha 1I depending on the organization unit of small-pore forming,and in the present invention, the inventors carried out an experimentrelated to pain by using the alpha 1G−/− transgenic mice so as tosuppress the function of the alpha 1G protein, one of the component ofalpha 1G T-type calcium channel. It has been known that mechanicalstimuli, which are acute pains, are controlled by spinal reflex, and theresponsiveness of the mechanical stimuli is proportionate with theintensity of the pains received by the peripheral organs. The alpha1G−/− mice do not have much difference from the normal mice in the pawwithdrawal and tail flick test (see FIG. 1). In addition, the result ofthe thermal pain response analysis using radiant heat in which spinalreflex and supraspinal mechanism is involved shows that the alpha 1G−/−mice does not have much difference from the normal mice, either inthermo-stimulus hyperalgesia caused by inflammation reaction orthermo-stimulus by infrared radiation (see FIG. 2). As mentioned above,deducing from the fact that the alpha 1G−/− mice reacts normally tothermal or mechanical stimuli, it can be known that the loss of thealpha 1G T-type calcium channel do not affect the development of theperipheral sensory organs, that is, the nerves that are involved inspinal reflection and inflammation reaction.

[0024] From the observation of the response of the internal painsinduced by acetic acid, the alpha 1G−/− mice show serious hyperalgesiato the visceral pain induced by acetic acid (see FIG. 3). It shows thatthe alpha 1G T-type calcium channel is selectively involved in thecontrol of visceral pain. In order to find out whether the alpha 1GT-type calcium channel that is involved in visceral pain controlfunctions in the peripheral sensory organs or in the part where thespine is connected to the brain and thalamus, the present inventorsinjected mibefradil, a T-type inhibitor, in the same spot of acetic acidinjection. The result was that there was analgesia to the visceral paincaused by the acetic acid (see FIG. 4). Mibefradil in this casesuppresses the T-type calcium channel only in the peripheral nervoussystem since it cannot pass through the brain barrier inside the body.

[0025] From the result, it can be deduced that the pain suppression ofthe alpha 1G T-type calcium channel only functions in the centralnervous system, not in the peripheral nervous system. It can also beknown that the other types of the T-type calcium channels, alpha 1H andalpha 1I calcium channels increase pain in the peripheral nervoussystem.

[0026] As described above, the T-type calcium channel functions whollydifferently in the peripheral nervous system and in the central nervoussystem concerning visceral pain control, and the T-type calcium channelis activated when the resting membrane potential lowers, thussuppressing the membrane potential from being low, which increase theactivity of the visceral pain sensory cells in the peripheral organs.This is supported by the fact that the T-type calcium channel functionsagainst hyperalgesia to mechanical or thermal stimuli induced byreducing agents. As for visceral pain, the dilation of intestinal cellsor outflow of reduced substrates from cells caused by necrosis isbrought along, so it is highly likely that hyperalgesia is alreadyincluded in the normal algesia.

[0027] The present invention also provides a visceral pain inhibitorthat includes a T-type calcium channel inhibitor as an effectiveingredient.

[0028] When a visceral pain inhibitor that includes a T-type calciumchannel inhibitor as an effective ingredient is injected into a body,the inhibitor reacts with alpha 1H and alpha 1I T-type calcium channel,which will eventually suppress visceral pain by suppressing thefunctions of the above-mentioned alpha 1H and alpha 1I T-type calciumchannel.

[0029] In the present invention, the T-type calcium channel inhibitor isselected from a group consisting of mibefradil and Ni²⁺.

[0030] Moreover, the present invention provides a method of screening avisceral pain inhibitor by investigating the suppression activation ofthe T-type calcium channel.

[0031] In the present invention, the suppression activity of the T-typecalcium channel of chemical materials or natural materials isinvestigated; the materials that have suppression activity to the T-typecalcium channel are selected; and among the selected materials, thematerial that has an analegesic effect only on the visceral pain inducedby acetic acid, et al. is found by carrying out experiments related painwith alpha 1G−/− mice and normal mice.

[0032] In accordance with the present invention, the T-type calciumchannel inhibitor has a suppression effect on visceral pain in a precisemechanism without any side effects, therefore, the selected materialfrom these T-type calcium channel inhibitors can be used as a visceralpain inhibitor.

EXAMPLES

[0033] Practical and presently preferred embodiments of the presentinvention are illustrative as shown in the following Examples.

[0034] However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Generation and Management of Alpha 1G−/− Transgenic Mice

[0035] <1-1> Generation of Alpha 1G−/− Transgenic Mice

[0036] The present inventors have produced transgenic mice that have thegenotype of alpha 1G−/− by using an embryo that has the genotype ofalpha 1G+/− (International Depository Authority: Korea Institute ofBioscience and Biotechnology Gene Bank, Accession No: KCTC 10086 BP).Particularly, the heterozygote transgenic mice having alpha 1G−/−genotype was produced by transplanting the embryo having alpha 1G+/−genotype to a surrogate mother, and the homozygote transgenic micehaving alpha 1G−/− genotype was produced by crossbreeding male andfemale of the above heterozygote transgenic mice.

[0037] <1-2> Management of Animal

[0038] All animals were allowed to access freely to food and water underthe environment of controlled temperature and humidity, and they werebred under the condition in which the daytime starts at 8 o'clock in a12 hour cycle of daytime and nighttime. All male and female F2 mice wereused in the experiments when they are 8-15 week old.

Example 2 Analysis of the Response to Mechanical Stimuli

[0039] In order to observe the response of the alpha 1G−/− mice tomechanical stimuli, the present inventors carried out a paw withdrawaltest and a tail flick test.

[0040] <2-1> Paw Withdrawal Test

[0041] Paw withdrawal test was based on that described by Mogil et al(Mogil et al., J. Neurosci., 1999, 19:RC25). Particularly, the alpha1G−/− mice were placed individually on a fine mesh metal floor andallowed to acclimate for at least 2 hr. The mechanical threshold wasmeasured using calibrated von Frey filaments (Stoelting) and was definedas the bending force, in grams, at which the mice withdraws its paw. Thefilament was applied from underneath the floor, through the mesh, to theplantar surface of the paw for each limb. The response score wasassessed as the total numbers of paw withdrawals in 10 consecutivetrials for each filament and the average value of the response was usedin the analysis.

[0042] <2-2> Tail Flick Test

[0043] The local pressure required to elicit tail flick was determinedusing von Frey filaments. The alpha 1G−/− mice were habituated in themice restrainer 30 minutes every day for 2 weeks. The bending force ofeach monofilament was applied locally to the tail resting on a table.Only flicking of the pressed tail was defined as a nociceptive response.The response score was assessed as the average of the total tailflicking number in 10 consecutive trials with an interval of 10 minbetween each filament application.

[0044] As a result, the alpha 1G−/− mice do not have any difference fromthe normal mice in responding to thermal or mechanical stimuli using vonFrey (FIG. 1)

Example 3 Responses to Radiant Heat and Hot Plate

[0045] The present inventors examined the thermal pain response by usingradiant heat assay (Hargreaves test) wherein the mechanism of spinalreflex and supraspine is involved.

[0046] <3-1> Paw Withdrawal Test

[0047] The present inventors measured hind-paw withdrawal latency byHargreaves' method (Hargreaves et al., Pain, 1988, 32:77-88) using anUgo Basile plantar test apparatus (Stoelting). Mice were placed in aPlexiglas box on an elevated glass plate and acclimated for 2 hr beforetesting. The tests were performed at low (20) and high (40) intensities.Response was defined as withdrawal of a paw when head turning and pawlicking were observed. The time was defined as the paw withdrawallatency. Five to ten minutes were allowed between each trial on bothhind-paws and 4 to 5 trials were averaged for each mice.

[0048] <3-2> Hot Plate Test

[0049] Thermal pain response was assessed using the hotplate test (Mogilet al., J. Neurosci., 1999, 19:RC25). For the hot-plate test, the micewas habituated for 2 days in a transparent testing box (14×14×20 cm)with a metal bottom. The mice was then placed on the box pre-heated tothe desired temperature in a thermo-regulated water bath, and the timewas recorded to the first hind-paw licking or jumping response (cut-offtime, 60 s).

[0050] As a result, the alpha 1G−/− mice did not have much differencefrom the normal mice in the response to thermal hyperalgesia caused byinflammation reaction or thermostimuli by infrared radiation (FIG. 2).Thus, it was confirmed that the loss of the alpha 1G T-type calciumchannel did not affect the development of the peripheral sensory organs,that is, the nerves that are involved in spinal reflection andinflammation reaction.

Example 4 Analysis of Visceral Pain Induced by Acetic Acid (WrithingTest)

[0051] The present inventors injected 0.6% acetic acid into theperitoneal cavity of mice to examine the acetic acid-induced visceralpain response. The visceral pain is elicited secondarily to a delayedinflammatory response and induced abdominal stretching and writhingbehavior (Gyires and Torma, Arch Int. Pharmacodyn. Ther., 1984,267:131-140). Mice were placed individually in a transparent home cage(24×18×12 cm) and allowed to acclimate for at least 60 min. Then, 0.6%acetic acid (5.0 mg/kg) was injected into the peritoneum, after whichthe mice was returned to the testing chamber. The number of abdominalstretches or writhing motions was counted for 20 min. All mice were usedonly once in this experiment.

[0052] As a result, the alpha 1G−/− mice showed severe hyperalgesia tothe visceral pain induced by acetic acid (FIG. 3), which means that thealpha 1G T-type calcium channel is involved selectively in controllingthe sense of visceral pain.

Example 5 Analysis of Visceral Pain Induced by Mibefradil

[0053] In order to find where the alpha 1G T-type calcium channelfunctions in relation to controlling visceral pain, whether theperipheral nerves or the part where the spine was connected to thalamusand brain, the T-type inhibitor, mibefradil, was injected intoperitoneum, the same spot where the acetic acid (the visceral paininducer for the normal mice) was injected.

[0054] Particularly, in order to find out how mibefradil, the T-typecalcium channel inhibitor functions, mibefreail was dissolved in 0.9% ofNaCl at the concentration of 5 mg/ml. The degree of writhing wasmeasured in a visceral pain inducing experiment 20 minutes after theinjection of the said mibefradil at the concentration of 1, 10 and 30mg/kg respectively in peritoneum of the mice.

[0055] As a result, the mibefradil induced analgesia to the visceralpain caused by acetic acid in the normal mice (FIG. 4).

INDUSTRIAL APPLICABILITY

[0056] As shown above, a method of the present invention can beeffectively used to suppress visceral pain by regulating T-type calciumchannel in a precise mechanism without any side effects.

[0057] Those skilled in the art will appreciate that the concepts andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

What is claimed is:
 1. A method for the suppression of visceral pain byregulating the T-type calcium channel.
 2. The method as set forth inclaim 1, wherein the T-type calcium channel is selected from a groupconsisting of alpha 1G, alpha 1H and alpha 1I T-type calcium channels.3. The method as set forth in claim 1, wherein the suppression ofvisceral pain is achieved by activating the function of alpha 1G T-typecalcium channel in the central nervous system.
 4. The method as setforth in claim 1, wherein the suppression of visceral pain is achievedby inhibiting the function of alpha 1H and alpha 1I T-type calciumchannels in the peripheral nervous system.
 5. A visceral pain inhibitorcontaining the T-type calcium channel inhibitor as an effectiveingredient.
 6. The visceral pain inhibitor as set forth in claim 5,wherein the T-type calcium channel is alpha 1H or alpha 1I T-typecalcium channel.
 7. The visceral pain inhibitor as set forth in claim 5,wherein the T-type calcium channel inhibitor is selected from a groupconsisting of mibefradil and Ni²⁺.
 8. A screening method of the visceralpain inhibitor by investigating the suppression activity of the T-typecalcium channel.